noscapine crosses the blood-brain barrier and inhibits

Noscapine Crosses the Blood-Brain Barrier and InhibitsGlioblastoma Growth

Jaren W. Landen,1 Vincent Hau,8

Mingshen Wang,2 Thomas Davis,8 Brian Ciliax,2

Bruce H. Wainer,3 Erwin G. Van Meir,4,5,6

Johnathan D. Glass,2 Harish C. Joshi,1 andDavid R. Archer7

Departments of 1Cell Biology, 2Neurology, 3Pathology,4Neurosurgery, 5Winship Cancer Institute, and6Hematology/Oncology, and 7the AFLAC Cancer Center and BloodDisorder Service, Emory University School of Medicine, Atlanta,Georgia; and 8University of Arizona, Department of Pharmacology,Tucson, Arizona

ABSTRACTThe opium alkaloid noscapine is a commonly used an-

titussive agent available in Europe, Asia, and South Amer-ica. Although the mechanism by which it suppresses cough-ing is currently unknown, it is presumed to involve thecentral nervous system. In addition to its antitussive action,noscapine also binds to tubulin and alters microtubule dy-namics in vitro and in vivo. In this study, we show thatnoscapine inhibits the proliferation of rat C6 glioma cells invitro (IC50 � 100 �M) and effectively crosses the blood-brainbarrier at rates similar to the ones found for agents such asmorphine and [Met]enkephalin that have potent centralnervous system activity (P < 0.05). Daily oral noscapinetreatment (300 mg/kg) administered to immunodeficientmice having stereotactically implanted rat C6 glioblasomainto the striatum revealed a significant reduction of tumorvolume (P < 0.05). This was achieved with no identifiabletoxicity to the duodenum, spleen, liver, or hematopoieticcells as determined by pathological microscopic examinationof these tissues and flow cytometry. Furthermore, noscapinetreatment resulted in little evidence of toxicity to dorsal rootganglia cultures as measured by inhibition of neurite out-growth and yielded no evidence of peripheral neuropathy inanimals. However, evidence of vasodilation was observed innoscapine-treated brain tissue. These unique properties ofnoscapine, including its ability to cross the blood-brain bar-

rier, interfere with microtubule dynamics, arrest tumor celldivision, reduce tumor growth, and minimally affect otherdividing tissues and peripheral nerves, warrant additionalinvestigation of its therapeutic potential.

INTRODUCTIONPatients diagnosed with glioblastoma (WHO grade IV)

have a median survival of 9–12 months despite surgical resec-tion, radiation therapy, and/or chemotherapy (1, 2). The infil-trative nature of astrocytic tumor growth rarely allows completesurgical resection, and more than 90% of tumors recur within 2cm of the primary tumor site. Postoperative radiotherapy pro-longs survival, but the prognosis is still less than 2 years.Intrinsic chemoresistance and poor penetrance of drugs throughthe blood-brain barrier remain significant challenges for thechemotherapeutic treatment of gliomas (3). Given the limitedefficacy of existing therapy, even when combined, there is aconsiderable need to direct research efforts to develop moreeffective treatments for brain tumors.

Malignant gliomas develop in part as a result of geneticmutation(s) in checkpoint genes resulting in deregulation of thecell cycle. Abrogation of the G1-S checkpoint is a frequent eventin the development of gliomas (4–7), implying a role for cyclin-dependent kinases cyclin-dependent kinase 4/6 and theircatalytic partners and D-type cyclins that are required for pro-gression through the G1-S phases of the cell cycle. The cyclin-dependent kinase/cyclin D complex is inhibited in response toDNA damage or inadequate cell growth by p16INK4 and CIP/KIP, resulting in the activation of the G1-S checkpoint and arrestof normal cells in G1 (8–13). Homozygous deletions of G1-Scheckpoint genes have been found in 41% of glioblastomas,suggesting that checkpoint mutations may contribute to theuncontrolled cell proliferation of glioblastoma (14). Other mu-tations have also been well documented including mutations inp53 (15) or in the retinoblastoma gene (16), each of which isfound in nearly one-half of all gliomas.

Our laboratory has identified a microtubule-interactingchemotherapeutic agent that overcomes many of the limitationsassociated with other tubulin-binding drugs. This agent, noscap-ine, is an antitussive opium alkaloid that lacks sedative, eu-phoric, analgesic, and respiratory depressant properties (17).The precise mechanism for the antitussive effects of noscapineis unknown, although it appears centrally mediated. Noscapinecan reduce electrically induced cough, characteristic of drugsaffecting the autonomic nervous system (18), and radiolabelednoscapine binds the central nervous system (19, 20). Coughsuppression was the only pronounced pharmacological effect ofnoscapine known for more than 30 years. In the last 5 years, wedemonstrated that noscapine: (a) binds to tubulin and alters itsconformation and assembly properties; (b) interferes with mi-crotubule dynamics both in vitro and in living cells; (c) arrestsa variety of mammalian cells in mitosis and targets them forapoptosis; and (d) inhibits growth of murine thymoma cells,

Received 2/24/04; revised 5/6/04; accepted 5/10/04.Grant support: NIH (H. Joshi, E. Van Meir, J. Glass), AmericanCancer Society (H. Joshi), the University Research Committee (H.Joshi), the Beat Leukemia Jill Andrews Fund (D. Archer), and BrainTumor Foundation for Children (D. Archer).The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.Note: H. Joshi and D. Archer contributed equally to this work.Requests for reprints: David R. Archer, Emory University School ofMedicine, Department of Pediatrics, Emory University School of Med-icine, 1462 Clifton Road, Room 466, Atlanta, GA 30322. Phone:(404) 727-1378; Fax: (404) 727-4859; E-mail: [email protected].

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human breast carcinoma, and melanoma cells in mice by induc-ing polyploidy and apoptosis (21–23). Furthermore, in contrastto other microtubule-interacting agents such as paclitaxel, no-codazole, and vinblastine, noscapine modifies microtubule dy-namics without affecting total tubulin polymer mass in reconsti-tuted systems and without altering the steady-state monomer/polymer equilibrium of microtubule assembly in cells (23).

In this study, we show that glioma cell treatment withnoscapine induces polyploidy. Noscapine-treated cells undergoexcessive DNA synthesis and atypical nuclear divisions in theabsence of cytokinesis, resulting in multinucleated cells. Wefurther show that noscapine crosses the blood-brain barrier andinhibits the growth of subcutaneous (s.c.) and intracranially(i.c.) implanted rat C6 glioma cells in immunocompromisedmice without apparent toxicity to organs with rapidly prolifer-ating tissues or induction of neurological symptoms.

MATERIALS AND METHODSMice and Cell Lines. Eight-week-old athymic female

mice (nu/nu) were purchased from the National Cancer Institute(Bethesda, MD). The rat C6 glioma cell line (American TypeCulture Collection) was maintained in DMEM supplementedwith 10% fetal bovine serum and passaged no more than 10times. Primary glial cells were isolated as follows. Cells fromthe mouse subventricular zone of C57BL/6 mice were dissectedunder a dissecting microscope, manually dissociated using aflame polished pipette, and grown in DMEM containing 20%fetal bovine serum. Glial cells used for experiments were iden-tified as cells that contain glial fibrillary acidic protein, anintermediate filament subunit found exclusively in glial cells.These cells did not express neuronal markers such as Tuj-1, anantibody that is specific for neuronal �-tubulin.

Cell Density Assay. Cell proliferation was determinedby the WST-1 tetrazolium salt assay (Boehringer Mannheim),which quantifies the amount of formazan dye formed whentetrazolium salt is cleaved by cellular mitochondrial enzymespresent in viable cells. Cells were plated at a density of 1 �103/well in 96-well microtiter plates in 0.2 ml of culture me-dium. Cells were allowed to adhere overnight and then incu-bated with 0, 0.1, 1, 2, 10, 50, 100, or 1000 �M noscapine (97%purity; Aldrich; 100� stock in DMSO) for 0, 12, 24, 48, 72, or96 h. The final concentration of DMSO in medium never ex-ceeded 1%. Five hours before the end of the specified incubationperiods, 50 �l of WST reagent were added to the cells. At theend of the incubation, cell density was estimated by measuringthe absorbance of the colored formazan reaction product at 450nm using a microtiter plate reader (Molecular Devices Ltd.,Crawley, West Essex, United Kingdom).

Tubulin, DNA, and Bromodeoxyuridine Staining. RatC6 glioma cells and primary glial cells were cultured on poly-L-ornithine-coated glass coverslips and allowed to adhere for24 h. To examine how noscapine affects microtubule morphol-ogy and DNA content, noscapine was dissolved in DMSO, andcells were then incubated at 37°C with 0, 25, 50, 250, 500, and1000 �M noscapine for 24, 48, 72, or 96 h. Cells were fixed inmethanol at �20°C for 5 min, incubated with anti-�-tubulinantibody (DM1A; 1:500 dilution; Amersham Biosciences) for2 h at room temperature, washed and incubated with a goatantimouse secondary antibody (1:200 dilution; Jackson) for 1 h

at room temperature, and visualized using immunofluorescence.To visualize DNA, tubulin-stained cells were further incubatedwith 20 �g/ml propidium iodide (Boehringer Mannheim) for 30min before washing with PBS. Cells were subsequentlymounted on coated glass slides and analyzed using confocalmicroscopy for microtubule morphology, DNA content, and thenumber of cells in mitosis (at least 100 cells were examined percondition). In a separate experiment, to examine whethernoscapine-treated cells go through multiple rounds of DNAsynthesis, cells plated as described above were treated withnoscapine for 72 h. After 24 h, 30 �M bromodeoxyuridine(BrdUrd; Sigma Chemical Co.) was added for the remaining 48 h.Cells were fixed and stained with anti-BrdUrd antibody (1:200dilution; Boehringer Mannheim) and stained with FITC-labeledsecondary antibody (1:100; Jackson), mounted on glass slides, andanalyzed for BrdUrd incorporation by confocal microscopy.

Flow Cytometric Analysis of Cell Cycle Status. Cellcycle status was determined by measuring cellular DNA contentafter staining with propidium iodide by flow cytometry (21).Cells (1 � 104) were plated on 10-cm dishes and incubated for24 h before the addition of 0, 50, 250, 500, or 1000 �M

noscapine in 1% DMSO for 0, 6, 12, 24, 48, 72, or 96 h.Staurosporine (100 nM), a well-known cytotoxic agent, was usedas a positive control. In an independent study, cells were treatedwith noscapine at the same dosages and durations specified asabove, washed three times with PBS at 37°C, and allowed torecover for 96 h in fresh medium without noscapine. Cells fromboth experiments were removed with trypsin, collected, washedtwice in ice-cold PBS, fixed overnight in 70% ethanol at�20°C, and centrifuged at 1000 � g for 10 min. Cells were thenresuspended in 30 �l of phosphate/citrate buffer [0.2 M

Na2HPO4/0.1 M citric acid (pH 7.5)] and incubated with pro-pidium iodide (20 �g/ml) and RNase A (20 �g/ml) in PBS for30 min. The propidium iodide fluorescence was measured usinga Becton Dickinson flow cytometer. Data were analyzed usingWinlist software (Verity Software House, Topsham, ME).

In Vitro Bovine Brain Microvessel Endothelial Cell As-say. Bovine brain microvessel endothelial cells were isolatedfrom the cerebral cortex as described previously (24) on poly-carbonate membrane filters. In brief, bovine brain microvesselendothelial cells were isolated from the gray matter of thebovine cerebral cortex by enzymatic digestion followed bysubsequent centrifugations and seeded into primary culture.Polycarbonate membranes (13 mm; pore size, 3.0 �m; diffusionarea, 0.636 cm2) were placed in tissue culture dishes (100 mm;Corning, Corning, NY) and coated with rat-tail collagen andbovine fibronectin (Sigma). Isolated brain microvessel endothe-lial cells were seeded onto the prepared tissue culture dishes ata density of 5 � 104 cells/cm2 in a culture medium consisting of45% MEM, 45% Ham’s F-12 nutrient mixture (Life Technolo-gies, Inc., Grand Island, NY), 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.4), 13 mM sodium bicar-bonate, 10% plasma-derived equine serum, 100 mg/ml heparin,100 mg/ml streptomycin, 100 mg/ml penicillin G, 50 mg/mlpolymyxin B, and 2.5 mg/ml amphotericin B (Sigma ChemicalCo.). The cells were cultured at 37°C with 5% CO2. Mediumwas replaced on the 3rd day after seeding, and then every 2 daysuntil confluent monolayers were formed (10–14 days). Conflu-

5188 Noscapine Crosses Blood-Brain Barrier, Inhibits Glioblastoma



ence was determined by inspecting the areas around the poly-carbonate membranes with an inverted microscope.

Bovine brain microvessel endothelial cell monolayers cul-tured on a polycarbonate membrane were placed in a Side-Bi-Side diffusion cell (Crown Glass Co., Somerville, NJ) contain-ing 3 ml of continuously stirred physiological assay buffer (122mM NaCl, 3.0 mM KCl, 1.2 mM MgSO4, 25 mM NaHCO3, 0.4mM K2HPO4, 1.4 mM CaCl2, 10 mM D-glucose, and 10 mM

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) on eachside at 37°C (Fig. 5). At time 0, noscapine (500 �M) was addedto the donor chamber, and 100-�l aliquots were removed fromthe receptor chamber at various time points (15, 30, 60, 90, and120 min) and stored for high-performance liquid chromatogra-phy (HPLC) analysis. An equal volume of assay buffer wasadded to replace the aliquots removed. [14C]sucrose (10.44Ci/mmol; NEN Research Products, Boston, MA), a moleculethat does not cross the membrane, served as a negative controland [Met]enkephalin (DPDPE; Tyr-Gly-Gly-Phe-Met), which isknown to permeate the blood-brain barrier, was used as apositive control. Background leakiness was monitored and cor-rected for by determining the levels of [14C]sucrose in thesamples via scintillation spectrometry (efficiency, 93% for[14C]sucrose; Beckman LS 50000 TD counter; Beckman Instru-ments Inc., Fullerton, CA). Passage of the test solute across thein vitro blood-brain barrier monolayer was determined by re-verse phase-HPLC as described previously (25). Reverse phase-HPLC values were obtained in moles and used to determinepermeability coefficients found by using the following equation:PC � X/(A � t � Cd) where PC is the permeability coefficient(cm/min), X is the amount of substance in moles in the receptorchamber at time t (min), A is a constant diffusion area (0.636cm2), and Cd is the concentration of the substance in the donorchamber (in mol cm�3).

In Vivo Tumorigenicity Assays. Immediately beforesurgery, rat C6 glioma cells were washed twice with PBS andplaced into serum-free DMEM medium. Thirty anesthetized8-week-old athymic nude mice received 1 � 103 rat C6 gliomacells stereotactically implanted into the left striatum (coordi-nates: anterior-posterior, �2.5; medial-lateral, �3.5; dorsal-ventral, �2 mm; from the Bregma). C6 glioma cells in a volumeof 2 �l were slowly injected over a time span of 15 min (forillustration, see Fig. 7A). Due to the age of the animals, theneedle easily penetrated the skull. Sham-operated animals (n �15) received an identical intracranial injection of serum-freemedium alone. After injection, the needle track was sealed withbone wax, and the incision was closed with Ethicon staples(Endo Surgery, Inc.). An independent group of 30 animalsreceived 1 � 106 rat C6 glioma cells in a volume of 0.2 ml s.c.into the right flank. Six days after s.c. injection when s.c. tumorswere palpable or six days after stereotactic injection, animalswere divided into two groups (n � 15/group). One group re-ceived noscapine hydrochloride by daily gavage [300 mg/kgdissolved in de-ionized water (dH2O; pH 4.5)], and the othergroup received the vehicle solution alone by gavage (dH2O; pH4.5). Tumor volumes were recorded biweekly for animals in thes.c. group. On day 21 (15 days of noscapine treatment), animalswith intracranial tumors were anesthetized with 4% chloralhydrate and then perfused intracardially with phosphate-buff-ered-saline followed by 2.5% gluteraldehyde and 2.5%

paraformaldehyde in phosphate buffer. At necropsy, the follow-ing tissues were taken for analysis: spleen; duodenum; liver;sciatic nerve; sural nerve; dorsal and ventral roots; and brain.Before perfusion, blood from the heart was taken for a completeblood count using a complete blood count instrument (CDCTechnologies, Oxford, CT), and bone marrow was removedfrom the right femur and tibia bones for WBC analysis using a25-gauge needle. Brain weight was obtained upon sacrifice forboth s.c. and intracranial tumor groups. On day 21 (15 days ofnoscapine treatment), animals in the s.c. tumor group (n � 30)received one final dose of noscapine (n � 15) or vehiclesolution (n � 15) 2 h before sacrifice to determine noscapineconcentrations in the brain at the reported half-life of noscapine(26). Animals in the s.c. group were sacrificed by cervicaldislocation, and the brains were retained for HLPC detection ofnoscapine as described below.

Determination of Noscapine Concentration in AnimalTissues by High-Performance Liquid Chromatography.Two h after the final noscapine administration (n � 15) orvehicle solution (n � 15), animals were anesthetized and thensacrificed by cervical dislocation. This timing was selectedbased on the reported half-life of noscapine (154 min; Ref. 27).Blood was collected before animal sacrifice directly from theheart and centrifuged, and plasma was removed and stored at�80°C for HPLC analysis of noscapine and its metabolites.Brains were removed without perfusion; meninges and exteriorblood vessels were dissected including the middle cerebralartery, the arteries forming the circle of Willis, superior saggitalsinus, and the transverse sinus. Dissected brains were homoge-nized and centrifuged to remove cell debris, and supernatantswere collected and stored at �80°C for HPLC analysis. HPLCanalyses were performed in a double-blind fashion according toa previously published method (28). In brief, samples wereanalyzed on a reverse-phase-HPLC system consisting of a WISP710B Autoinjector, two model 6000A Solvent Delivery Pumps,Automated Gradient Controller (Waters Associates, Milford,MA), LC-65T Detector/Oven (210 nm; Perkin-Elmer, Norwalk,CT), 3390A Integrator (Hewlett-Packard Co., Avondale, PA),and a 218TP54 column (4.6 � 250 mm; Vydac, Hesperia, CA).Samples were eluted using a linear gradient of acetonitrileagainst 0.1 M NaH2PO4 buffer (pH 2.4). The flow rate wasmaintained at 1.5 ml/min, and the column temperature at 40°C.The capacity factor was defined as follows: k � (tr � to)/towhere tr is the retention time of the retained peak and to is theretention time of an unretained peak.

Image Analysis. Brains from animals that received i.c.injections were fixed by perfusion as described above andsectioned into 1-mm-thick slices using a brain matrix (for illus-tration, see Fig. 8B; Ted Pella, Redding, CA). Each 1-mm-thicksection was then embedded in paraffin blocks maintainingproper orientation so that the anterior-most side would be sec-tioned first. A single 5-�m section was then cut from each blockand stained with H&E for three-dimensional reconstruction.Slides were coded, and each section was captured at an identicalmagnification using a digital camera (SPOT camera; DiagnosticInstruments Inc., Sterling Heights, MI). Tumor cells were iden-tified because they stained with greater intensity than surround-ing normal striatal cells as shown in Fig. 7A (arrow). Thedifferences in staining intensity were detected using AIS image

5189Clinical Cancer Research



analysis software (Imaging Research, Inc., St. Catherine’sHeights, Ontario, Canada). The system was calibrated using amicrometer and selecting the appropriate number of pixels equalto 1 mm. Tumor cross-sectional area was determined by usingan auto-sampling tool, which bases its selection on stainingintensity. The autoscan tool uses an algorithm to select the targetregion based on H&E-staining intensity differences between theselected target gray value and background gray value identified.The autoscan feature searches in concentric circles seeking acontinuous boundary. This boundary is defined by an averagethreshold level of staining intensity that circumscribes the targetarea pixel by pixel, radiating outward from the first darklystained pixel selected. Adjacent tumor regions in the samesection were identified as separate targets. This procedure en-abled us to quantify infiltrative tumor regions consisting ofnormal and tumor tissue within a boundary because normaltissue had lower than threshold-staining intensity and thus wasexcluded from the cross-sectional tumor area measurements.The autoscan feature also excluded open spaces such as vessellumens (for illustration, refer to Fig. 7, C and D). Tumor regionswere verified by manual microscopic inspection by a pathologist(B. H. W.). The three-dimensional tumor volume was computedusing cross-sectional tumor area � 1 mm (the distance between5-�m sections). If more than one tumor target site was presentin a cross-sectional tumor area, targets were added for thatsection. The blind code was broken after all of the sections hadbeen scanned and tumor volumes obtained.

Toxicity Evaluation. After animal perfusion and sacri-fice on day 21, liver, duodenum, and spleen were sectioned,stained with H&E, and analyzed by two pathologists (B. H. W.and D. L. D.) for microscopic evaluation. Bone marrow wasremoved before fixation from the femur and tibia bones andanalyzed by flow cytometry following antibody lineage-mark-ers: CD3 (T cells); B220 (B cells); MAC-1 (macrophages); andGr-1 (granulocytes; PharMingen, San Diego, CA). Cells werealso incubated with 20 �g/ml propidium iodide to determine thepercentage of dead cells. Sciatic nerve, dorsal root ganglion,dorsal and ventral roots, and sural nerves were removed fromfour animals from each group, embedded in plastic, cut at 1 �m,and stained with 0.5% toluidine blue for microscopic analyses.Sections were evaluated blindly for evidence of sensory andmotor neuropathies by a neuropathologist (J. D. G.).

Dorsal Root Ganglion Cultures and Evaluation of Neu-ropathy. Dorsal root ganglion neurons were cultured as de-scribed previously (29). In brief, dorsal root ganglions weredissected from newborn mice. Ganglia were transferred intoL-15 medium (Life Technologies, Inc.), separated from rootsand connective tissue sheaths, pooled, dissociated, and washedtwice with PBS (pH 7.4). Dorsal root ganglions were then plated(five per dish) in MEM supplemented with 1% N2 (Life Tech-nologies, Inc.), 10 ng/ml 7S nerve growth factor (Sigma), and1.4 nM L-glutamine (Sigma) and incubated at 37°C in a 5%carbon dioxide atmosphere. Next, cultures were permitted tomature for 5 days to allow a lush halo of neurites around theexplants to develop. Neuritic extensions were allowed to pro-ceed to evaluate the effect of noscapine on established neuritesas opposed to the effect on primary neurite outgrowth. After 5days in the culture medium, the medium was changed and 25,50, or 250 �M noscapine or DMSO vehicle solution alone (final

concentration 1%) was added. Cultures were monitored andimaged on 0, 4, 8, and 10 days post noscapine treatment. Thediameter of the circular halo of neurites was measured on theinitial day of noscapine exposure (day 0), and on days 4, 8, and10. Axonal survival was quantified by the longest remainingaxon and the area of the remaining dorsal root ganglion halo(Fig. 10B). The axonal length was measured from the center ofthe halo to the visible distal ends of the axon in the periphery ofthe halo. Halo areas were calculated by tracing the outsidecircumference of the remaining culture halo. Because there wasvariability in the physical characteristics of individual cultures,each dorsal root ganglion served as its own control by normal-izing data at days 4, 8, and 10 to the condition before noscapineexposure. Data were analyzed as a percentage change from day0 before noscapine treatment. Normalized data were then ex-

Fig. 1 Dose and time effects of noscapine on normal and tumor glialcells in vitro. A, noscapine inhibits cell viability of rat C6 glioma in vitroin a dose-dependent manner. Noscapine exposure (250 �M; arrow) for72 h inhibited cell viability of rat C6 glioma cells (F) by 50%. Primaryglial cells (�) were almost one-half as sensitive to noscapine (IC50 �500 �M). B, kinetics of noscapine treatment on cell viability. Incubationwith 250 �M noscapine for 24, 48, 72, and 96 h. F, rat C6 glioma cells;�, primary glial cells.




amined for statistical significance by ANOVA, with post-testcorrection for multiple comparisons (29).

RESULTSNoscapine Inhibits Rat C6 Glioma Cell Proliferation.

To determine whether noscapine could inhibit glioma cell growthin vitro, we chose the aggressive and rapidly dividing rat C6 gliomacell line. Using the tetrazolium salt (WST-1) cell viability assay(see “Materials and Methods”), we generated a dose-responsecurve by incubating cultures of rat C6 glioma cells and primarymouse glial cells (as normal tissue control) with noscapine for 72 hand observed a dose-dependent inhibition of cell viability (Fig. 1A).Noscapine inhibited the viability of rat C6 glioma cells with an IC50

of 250 �M at 72 h. Primary glial cells were less sensitive, having anIC50 of 500 �M (at 72 h). Manual counts of cell numbers verifiedthis finding (not shown). Because 250 �M noscapine exposure

resulted in a significant inhibition of glioma cells while primarycells were nearly unaffected, we selected this dose to examine howthis concentration affects cell viability over time (24–96 h; Fig.1B). We conclude that 250 �M noscapine exposure for 72 h is anoptimal dose to inhibit C6 glioma cell viability without signifi-cantly reducing primary cell viability.

Noscapine Exposure Causes Abnormal S-Phase Re-entry and Results in Excessive DNA Accumulation. Themechanism of the decreased cell viability observed in C6 cellsexposed to 250 �M noscapine for 72 h was evaluated by ana-lyzing the cell cycle distribution by flow cytometry with pro-pidium iodide (Fig. 2). Cultures of C6 cells or primary murineglial cells were treated with noscapine, fixed, and stained withthe DNA intercalating fluorescent dye propidium iodide. Un-treated C6 cells (Fig. 2C) and primary murine glial cells (Fig.2A) exposed to vehicle alone (1% DMSO) had normal cell cycle

Fig. 2 Noscapine induces polyploidy in rat C6 glioma cells. Rat C6 glioma cells and murine primary glial cells were treated with 250 �M noscapinein DMSO or vehicle solution alone (DMSO) for 72 h and then fixed and stained with the DNA-binding fluorescent dye propidium iodide (PI) andwith BrdUrd (BrdU). A, 72% of vehicle-treated primary glial cells contain 2N DNA content (peak in green), and 14% of cells contain 4N DNA content(peak in blue). Approximately 50% of primary cells incorporated BrdUrd (inset in green) compared with the nuclear staining shown in red. B, 24%of primary cells exposed to 250 �M noscapine for 72 h had 2N DNA content. Note the increase in the number of cells (37%) with 4N DNA contentafter noscapine exposure (peak in blue). Fifty percent of noscapine-treated primary cells incorporated BrdUrd (insets). C, vehicle-treated glioblastomacells had a DNA content similar to vehicle-treated primary cells with 69 and 18% of cells containing 2N and 4N DNA content, respectively.BrdUrd-positive vehicle-treated glioma cells were observed in approximately 60% of cells. D, noscapine-treated glioma cells contained 1% of cellswith 2N DNA content and 9% of cells with 4N DNA content. Note the position of the peak showing cells with abnormal �8N-16N DNA content(yellow peak). This phenomenon is not seen in untreated C6 glioma cells (C) or in primary glial cells (A and B). Bar � 5.0 �m.




profiles with approximately 70% of cells in G0-G1 phase with2N DNA content (Fig. 2, left peaks in green) and 16% of cellsin G2-M phase containing 4N DNA content (Fig. 2, right peaksin blue). In primary glial cells, noscapine exposure (250 �M for72 h) resulted in a decrease to 24% of G0-G1 phase cells with 2NDNA content and an increase of cells in G2-M with 4N DNAcontent to 37% (Fig. 2B). In contrast, only 1% of rat C6 gliomacells contained 2N DNA content, and 9% of cells contained 4NDNA (Fig. 2D, blue peak) content after 72 h of continuousnoscapine exposure. In addition, 44% of cells contained 8N-16NDNA (Fig. 2D, yellow peak). Primary glial cells (Fig. 2B) didnot accumulate enhanced DNA content at identical doses, butrather, we observed reversible mitotic arrest after continuousnoscapine exposure for up to 6 h (not shown). Flow cytometricdata suggest that noscapine exposure causes 4N DNA accumu-lation in primary glial cells, suggesting G2-M arrest; whereas itresults in 8N-16N DNA accumulation in C6 cells, suggestingcontinuous DNA synthesis.

To determine whether C6 cells exposed to noscapine ac-

cumulate DNA by inappropriately reentering multiple rounds ofS phase, immunofluorescence was performed to detect BrdUrdincorporation (Fig. 2, inset in green), a thymidine analog incor-porated into cells during DNA synthesis (S phase). Nuclearstaining with propidium iodide (Fig. 2, inset in red) was used todetect all nuclei. Anti-BrdUrd staining was apparent in about50% of the primary glial cells treated with vehicle alone (Fig.2A, inset in green) or with noscapine (Fig. 2B, inset in green).Anti-BrdUrd staining was also present in approximately 50% ofC6 cells treated with vehicle (Fig. 2C, inset in green), indicatingthat cell division occurred during the course of the experiment.In contrast, manual cell counts revealed that 97% of C6 cellsincubated with noscapine for 72 h incorporated BrdUrd underthese conditions, indicating that most cells were in S phase.These cells showed multiple discrete BrdUrd-positive micro-nuclei (Fig. 2D, inset in green and nuclear staining in red) andhad DNA content between 8N and 16N, suggesting that noscap-ine-treated cells were undergoing multiple rounds of DNA rep-lication in the absence of cytokinesis resulting in cells with

Fig. 3 Noscapine exposure results in multinucleated cells with abnormal mitotic figures in rat C6 glioma cells. Double-labeling immunofluorescenceis shown with an anti-�-tubulin antibody that stains microtubules in green and propidium iodide nuclear staining that is shown in red. Noscapine-treated rat C6 glioma cells have large, abnormal, multilobed nuclei and intact microtubule arrays (E); and mitotic figures, when observed, wereabnormal with multiple microtubule asters and misaligned chromosomes (F). These effects were not apparent in noscapine-treated primary glial cells(B and C) or in vehicle-treated primary glia (A) or vehicle-treated rat C6 glioma cells (D). Note that noscapine exposure did not alter microtubulemorphology in primary glial cells (B) or in rat C6 glioma cells (E). Bar � 5.0 �m.




multiple nuclei. These features were not seen in primary cells(Fig. 2, A and B) or in vehicle-treated glioma cells (Fig. 2C).

Noscapine treatment does not perturb the morphology ofmicrotubule arrays and results in multinucleated glioma cellswith abnormal mitoses. To examine microtubule morphology,cellular microtubule arrays were observed by immunofluores-cence (Fig. 3). We used an antibody against �-tubulin (Fig. 3,green) to stain microtubules and the DNA-specific stain, pro-pidium iodide (Fig. 3, red), to stain chromosomes. In vehicle-treated C6 glioma and primary glial cells, microtubule arrayswere localized throughout the cytoplasm of interphase cells(Fig. 3, A and D). Microtubule arrays in vehicle-treated cellsappeared similar to those in untreated control cells (not shown),indicating that the vehicle solution (DMSO final concentrationwas less than 1% in cell medium) had no effect on microtubulearrays. Microtubule morphology of interphase primary glialcells in the presence or absence of 250 �M noscapine for 72 hwas similar (Fig. 3, compare A and B). Mitotic figures of normalglial cells were not visibly affected by noscapine, as shown bychromosomes properly aligned on the metaphase (Fig. 3C). Incontrast, as already observed in Fig. 2, C6 glioma cells treatedwith noscapine revealed large, abnormal, multiple nuclei (Fig.3E). Their microtubule structure was similar to that of vehicle-treated cells (Fig. 3, compare E with D). However, the spindlestructure of mitoses observed in noscapine-treated glioma cellswere abnormal with misaligned chromosomes and multiple mi-crotubule asters (Fig. 3, compare F with C). These data suggestthat noscapine treatment causes abnormal mitoses and accumu-lation of micro-nuclei in rat C6 glioma cells, whereas cultures ofprimary glial cells do not accumulate DNA, and normal mitosesare observed when exposed to noscapine.

Noscapine Exposure Causes Increased Mitotic Arrest.To quantify the number of cells in M phase of mitosis in thepresence or absence of noscapine, cells were treated with 250�M noscapine for incubation periods ranging from 0 to 96 h andthen fixed and stained with an anti-�-tubulin antibody andpropidium iodide, and the number of cells in mitosis counted(Fig. 4). Noscapine treatment (250 �M) resulted in an increase inthe number of mitotic figures observed in a time-dependentmanner up to 24 h. Mitotic figures were observed in 68% ofprimary glial cell cultures at 24 h. This is a significant increasefrom approximately 5% observed in vehicle-treated cells at anytime point. In contrast, only 36% of noscapine-treated C6 cellswere observed in mitosis at 24 h. The number of cells observedarrested in mitosis after 48, 72, or 96 h of noscapine exposuredecreased in a time-dependent manner. This might be due todrug inactivation/degradation (reported half-life ranges from 1.7to 4.5 h; Ref. 27). These data are compatible with the hypothesisthat noscapine arrests normal and tumoral glial cells in M phaseof the cell cycle. Although noscapine-treated glioma cells mayinitially arrest in mitosis, they overcome the M-phase block andreenter multiple rounds of DNA synthesis.

Noscapine Crosses the Blood-Brain Barrier. To exam-ine the ability of noscapine to cross the blood-brain barrier, we useda well-characterized in vitro assay (illustrated in Fig. 5A). The rateat which noscapine can cross a layer of cultured brain microvas-cular endothelial cells separating a donor and receiver chamber wasdetermined and compared with known permeant (morphine and[Met]enkephalin) and nonpermeant ([14C]sucrose) molecules.

Noscapine was deposited at a concentration of 500 �M in the donorchamber, and aliquots were removed from the receiver chamber at15, 30, 60, 90, and 120 min. The noscapine concentration wasdetermined by HPLC, and the apparent permeability coefficient (incm/min) calculated as described in “Materials and Methods.” Pas-sage of noscapine across the barrier was determined by the con-centration detected by HPLC in the donor chamber compared withthe receiver chamber. Noscapine was detected in the receiverchamber at a concentration of 10.21 �M (0.2%) after 15 min thatincreased to 96.6 �M after 120 min (19%; Fig. 5B). Noscapine wasfound to cross the simulated blood-brain barrier with a permeabilitycoefficient of 21.7 � 10�4 cm/min. This rate is 31.8% moreefficient than morphine (14.8 � 10�4 cm/min), an opiate known topossess lipophilic character and permeate the barrier (Fig. 5B; P �0.05; Student’s t test), and similar to the positive control, [Met]en-kephalin (DPDPE; 24.24 � 10�4 cm/min). Although this modeldoes not account for drug metabolism that occurs in vivo, it pro-vides evidence that noscapine efficiently crosses the blood-brainbarrier compared with other agents known to permeate well.

Next, we determined whether noscapine was transportedacross the blood-brain barrier in vivo. We homogenized and cen-trifuged whole brains of noscapine-treated animals and then deter-mined noscapine concentration by HPLC in supernatants. Theaverage noscapine concentration obtained from brain homogenatesof noscapine-treated animals was 18.2 3.7 �M (SD). Theanimals used for this study were not perfused, so the values ob-tained include the amount of noscapine present in brain tissue andthe vascular network. These results are also consistent with theradioactive data describing noscapine accumulation in rat brain

Fig. 4 Measurement of mitotic figures in glioblastoma and primaryglial cells. Noscapine preferentially arrests glioma cells in mitosis. Atime course of mitotic index is shown after noscapine exposure for 12,24, 48, 72, and 96 h. Noscapine treatment (250 �M) resulted in anincrease in the number of mitotic figures observed in a time-dependentmanner up to 24 h. Mitotic figures were observed in 68% of noscapine-treated primary glial cell cultures at 24 h (�). In contrast, only 36% ofnoscapine-treated rat C6 glioblastoma cells were observed in mitosis at24 h (F). The number of cells observed arrested in mitosis after 48, 72,or 96 h of noscapine exposure decreased in a time-dependent mannerwith 42% primary glial cells and 20% glioma cells arrested at 48 h and12 and 5%, respectively, at 72 h. At 96 h, approximately 5% of both celltypes were observed in mitosis. In the absence of noscapine, the numberof cells in mitosis at any given time was approximately 5% (� and E,primary glia and rat C6 glioma, respectively).




(19). Our in vitro and in vivo results suggest that noscapine iscapable of efficiently crossing the blood-brain barrier.

Noscapine Inhibits Rat C6 Glioma Growth in Vivo. Toexamine the ability of noscapine to inhibit the growth of tumorsin vivo, we first injected rat C6 glioma cells s.c. into immuno-deficient mice. 15 days of noscapine administration beginning 6days after s.c. implantation of 1 � 106 C6 glioma cells signif-icantly inhibited tumor growth (Fig. 6, P � 0.01; Student’s ttest). On day 21, s.c. tumor volume was reduced by 60% in thenoscapine-treated group compared with the vehicle-treatedgroup (1510 237 and 3739 586 mm3, respectively; n � 12and 11 respectively). As a result of the large s.c. tumors, overallanimal body weight significantly increased in untreated animals(mean weight at the time of sacrifice of vehicle-treated animalswas 28.61 2.02 g, and the mean weight of noscapine-treatedanimals was 23.36 1.49 g; weight SE; P � 0.01; Student’st test). The weight of the resected tumors could partially accountfor this observation. The average s.c. tumor mass taken from

vehicle-treated animals was 2.56 1.62 g compared with0.79 0.44 g removed from noscapine-treated animals.

To demonstrate that noscapine can treat gliomas in theirorthotopic brain location despite the blood-brain barrier, we nextinjected 1 � 103 C6 cells i.c. into the striatum of immunocom-promised mice (n � 30). Noscapine daily by gavage (300mg/kg; n � 15) or vehicle solution alone (n � 15) was admin-istered to animals for 15 days beginning on day 6, and animalswere euthanized 21 days after tumor implantation. The dailynoscapine dosage of 300 mg/kg (corresponding to approxi-mately six times the in vitro concentration) was chosen based onnoscapine solubility and the favorable IC50 of 250 �M at 72 h.Intracranial tumor volumes were analyzed as follows: perfusedbrains were cut into 1-mm-thick sections (Fig. 7B) and embed-ded into paraffin. The first 5-�m section from each block wascut, stained (H&E), and examined for cross-sectional tumorarea. Representative brain sections of mice treated or not withnoscapine are shown in Fig. 8, A and B, respectively. The

Fig. 5 Measurement of noscap-ine diffusion across an in vitroblood-brain barrier model. A,schematic of in vitro blood-brainbarrier model depicting the donorchamber in which noscapine wasdirectly added (right chamber)and the receiver chamber (leftchamber) from which aliquotswere taken at 15, 30, 60, 90, and120 min and noscapine concen-tration was determined. Cham-bers were separated by a layer ofbovine brain microvessel endo-thelial cells (BBMEC) coculturedwith astrocytes on a polycarbon-ate membrane. B, permeabilitycoefficients (P.C. value) derivedfrom measurements taken fromthe receiver chamber aliquotsand analyzed for noscapine con-centration by HPLC analysis.Permeability coefficient valuesof noscapine compared with anopiate, morphine, or [Met]en-kephalin, a compound known topermeate the barrier well, areshown in the table. Permeabilityof the negative control, [14C]su-crose was subtracted from eachcondition.




sections revealed extensive migration of tumor cells (identifiedby their darker staining) in the injected hemisphere in bothgroups (Fig. 8, A and B, and insets E and F). Brains of untreatedanimals showed a dense twirling pattern of tumor cells that

largely replaced normal brain tissue in the tumor center whileinfiltrating extensively normal brain at the periphery (Fig. 8E).Lumens of large blood vessels were much smaller than in thecontralateral tumor-free hemisphere, possibly suggesting com-pression by interstitial pressure (Fig. 8E). Brains of noscapine-treated animals showed clearly reduced numbers of tumor cells.Tumor cells in noscapine-treated tissue extensively infiltratedthe normal brain and tended to cluster around blood vesselswithout altering lumen size. In some cases, small areas ofhemocyanin were noted, an observation that usually reflectssubsided hemorrhage. Noscapine may have induced death ofsome rapidly proliferating endothelial cells in tumor vascula-ture, leading to vessel leakage. To try to quantify the differencein tumor burden between both groups while accounting for theintermixing of normal and tumor cells, we used digital imaging.Cross-sectional tumor area was determined for each 5-�m sliceand representative striatal sections from vehicle- and noscapine-treated animals are shown (Fig. 8, C and D, blue). Usingthree-dimensional image analysis reconstruction of brain tumorvolume, we found that 15 days of noscapine treatment signifi-cantly inhibited intracranial brain tumor growth by 78% (Fig.8G, P � 0.01; Student’s t test). In addition, we found a trendtoward increased brain weight in vehicle-treated animals com-pared with noscapine-treated animals receiving intracranial tu-

Fig. 6 Time course of s.c. rat C6 glioma tumor growth. Palpabletumors were established 6 days after injecting 1 � 106 rat C6 gliomacells s.c. in mice. Mice were treated beginning on day 6 with 300 mg/kgnoscapine in acidified water by gavage daily for 15 days (F), whereasvehicle-treated animals received acidified water alone by gavage (�).Tumor volume shown is SE. Day 21, P � 0.01 (Student’s t test).

Fig. 7 Rat C6 glioma cellswere stereotactically implantedinto the striatum. A, striatal sec-tion depicting india ink ster-eotactically injected using thecoordinates shown. Rat C6 gli-oma cells (1 � 103) in serum-free DMEM (vehicle solution)or vehicle solution alone wereprecisely implanted into thestriatum of nude mice using a10-�l Hamilton syringe. AP,anterior-posterior; ML, medial-lateral; DV, dorsal-ventral. B,after 15 days of 300 mg/kgnoscapine treatment in vehiclesolution (dH2O; pH 4.5) or ve-hicle solution alone adminis-tered by gavage, animals wereeuthanized and perfused, andbrains were removed and cutinto 1-mm-thick macrosectionsas shown here.




mors (median brain weight, 0.50 0.06 and 0.42 0.09 g foruntreated and noscapine-treated, respectively, SE). This issuggestive of increased brain density as a result of the tumortissue. The striking inhibition of tumorigenicity observed in the

intracranial tumor models suggest that noscapine may be effec-tive for the management of some types of gliomas.

Toxicity Evaluation. Given the efficiency of noscapineto reduce tumor growth, the next concern was to examine its

Fig. 8 Noscapine significantly in-hibits the growth of an intracranialglioma model. C6 glioma cells(103) were stereotactically injectedinto the striatum of nude mice.Animals received noscapine or ve-hicle solution daily beginning onday 6 and were sacrificed on day21. A-B, macrosections were inde-pendently embedded in paraffinblocks, and the first 5-�m sectionwas cut and stained with H&E todetermine the cross-sectional tu-mor area for each section. Repre-sentative H&E-stained striatal5-�m sections of vehicle-treated(A) and noscapine-treated (B) ani-mals. Cross-sectional area was de-termined using an autoscan toolthat uses an algorithm to select thetarget region based on intensitydifference between target (darklystained tumor cells) and back-ground (light pink regions) omit-ting normal tissue and emptyspaces (described in detail in “Ma-terials and Methods”). The tumorregions obtained for representativeuntreated (C) and noscapine (D)sections using the autoscan toolare depicted in blue. E and F, amagnification of the region shownin blue for an untreated (E) andnoscapine-treated (F) animal. G,daily noscapine treatment for 15days resulted in a significant 78%inhibition of rat C6 glioma in-tracranial tumor growth (o) whencompared with vehicle-treated an-imals (f). �, P � 0.05 (Student’st test). Bar � 150 �m.




potential toxicity in a variety of tissues. Extending our previousfindings (22), we show that noscapine had no apparent systemictoxicity, even in animals carrying a heavy tumor burden andbrain tumors known to cause overall anergy (30). Treated ani-mals did not show any signs of behavioral or neurologicaldeficit and were equally active as untreated animals and gainedweight. However, unexplained blood vessel dilation in noscap-ine-treated brain tissue was observed in both the tumor-infil-trated and contralateral hemispheres compared with untreatedtissue (Fig. 8F).

Hematological toxicity was absent as determined by com-plete blood count (Fig. 9B). No significant toxic side effectscould be detected by histopathology in sites of rapidly dividing

tissues such as spleen, duodenum, and liver as revealed byhistopathology (Fig. 9, A–C). Given that the principal toxicity ofexisting microtubule-targeting agents is peripheral neuropathy,we examined peripheral motor and sensory nerves for evidenceof neuropathy. We did not find evidence of either tubulo-vessicular accumulations, as may be seen with impaired axonaltransport, or axonal degeneration in either sensory or motor fibers(Fig. 10A). These types of pathological changes have been reportedwith other agents that disrupt microtubule function. The absence ofsuch pathology suggests that noscapine may be less toxic to pe-ripheral nerves than other reported tubulin-binding agents.

To further evaluate any potential toxic effects of noscapineon peripheral nerves, we examined dorsal root ganglion cultures

Fig. 9 Daily noscapine treat-ment (300 mg/kg) does not in-duce pathological abnormalitiesin tissues with frequent cellproliferation. A, representativemicrographs showing H&E-stained 10-�m-thick sections ofduodenum, spleen, and liverfrom noscapine-treated andvehicle-treated groups of mice.No histopathological differ-ences were noted in these tis-sues. B, complete blood countanalysis of tumor-bearing micetreated (red bars) and untreated(blue bars) with noscapine. Nosignificant differences couldbe detected between the twogroups in the WBC count(WBC), lymphocytes (LY), RBCcount (RBC), hemoglobin con-centration (Hb), mean corpus-cular volume (MCV), meancorpuscular hemoglobin (MCH),mean corpuscular hemoglobinconcentration (MCHC), meanplatelet volume (MPV), neutro-phils (NE), monocytes (MO),eosinophils (EO), and basophils(BA). C, comparison of distinctWBC populations found in thebone marrow of noscapine-treated or untreated animals (an-alyzed by flow cytometry usingspecific lineage markers). No dif-ference could be detected in thepopulation of B cells, T cells,macrophages (Mac), and granu-locytes (Gran) between the no-scapine-treated (o) and thevehicle-treated f groups. Bar �150 �m.




Fig. 10 Daily oral noscapinetreatment results in minimal ev-idence of peripheral neuropa-thy. A, representative ventralroot sections stained with tolu-idine blue from vehicle- andnoscapine-treated animals. B,cultured dorsal root ganglioncells (DRG) in the absence andpresence of noscapine. Totalaxonal length and the dorsalroot ganglion cell halo areawere the quantitative parame-ters used to measure neurotox-icity. Dorsal root ganglion cellswere cultured for 5 days andthen incubated with noscapinefor up to 10 days. Percentchange in axonal length (C) andpercent change of dorsal rootganglion cell halo area (D) after0 �M (f), 25 �M (o), 50 �M

(1), or 250 �M (2) noscapinetreatment for 4, 8, or 10 daysare shown.




in the presence or absence of noscapine (Fig. 10, B–D). Culturesexposed to 25 and 50 �M noscapine for 10 days demonstratedslowing of growth rate compared with control cultures (Fig. 10,C and D). Exposure to 250 �M, however, caused axonal degener-ation as measured by progressive reduction in axonal length andreduction of dorsal root ganglion area. These types of changes aretypical of those seen with exposure to vincristine or Taxol (31, 32).

DISCUSSIONAntineoplastic agents that interact with microtubules rep-

resent an important group of drugs that disrupt mitosis andparticularly mitotic spindle activity by interfering with micro-tubule dynamics. Microtubule-targeting drugs currently in useeither promote excessive stability of microtubules, such as thetaxane family, or induce depolymerization of microtubules likethe Vinca alkaloids (33). Our prior results suggest that the mostprominent effect of noscapine is on microtubule dynamics,significantly enhancing the percentage of time microtubulesspend idle or in a paused state (22). In this study, we show thatnoscapine significantly reduces the viability of rat C6 gliomacells at doses that do not induce death in primary mouse glialcells. We cannot exclude that the dose calculations and com-parisons drawn between the control mouse glial cells and rat C6glioma cells could vary slightly given potential species-specificsensitivities to drugs. A significantly greater number of primaryglial cells compared with glioma cells arrested in mitosis afternoscapine treatment. Mitotic C6 glioma cells but not normalglial cells became polyploid after nuclear endoreplication. Mi-totic cells showed abnormal spindle formation with excessiveand misaligned chromosomes leading to multinucleated cells.Primary cells arrested in G2-M without enhanced DNA accu-mulation, whereas treated glioblastoma cells escaped mitoticarrest and accumulated up to 16N DNA content by enteringsuccessive rounds of DNA synthesis in the absence of celldivision. Our data suggest that C6 glioma cells may have defi-cient G1-S and/or mitotic checkpoints, accounting for the en-hanced DNA content and abnormal mitoses observed.

Because cell cycle checkpoint mechanisms in tumor cellsare frequently faulty (34–36), cancer cells may be more suscep-tible than normal cells to noscapine. Our data support thehypothesis that transformed cells proceed improperly throughthe cell cycle resulting in abnormal mitoses and ultimatelyundergo cell death. Evidence of apoptosis was not observed inthese experiments. Abrogation of the G1-S checkpoint is afrequent event in the development of gliomas (4–6), and this isknown to cause failure of arrest of division (continued replica-tion) in response to treatment with microtubule-targeting drugs(37). p53 interacts with the centrosome and regulates centro-some duplication (38). p53 prevents cell cycle progression whenspindle assembly is blocked by antimicrotubule agents (39), andabnormal centrosome amplification and unbalanced chromo-some segregation are observed in p53 null fibroblasts (40).Consistent with these findings, p53 was found to prevent hy-perploidy in human glioma cells exposed to nocodazole (41).Whether a similar mechanism operated in C6 glioma cells inresponse to noscapine is unclear. C6 glioma cells have beenreported to contain wild-type TP53 alleles, but the p53 pathway

might be defective through alternative means such as p14ARFdeletion, a common feature in gliomas (42, 43, 44).

Our observation that noscapine crosses an experimentalblood-brain barrier efficiently led us to study whether noscapinecould inhibit the tumorigenicity of the rapidly dividing rat C6glioma cell line in vivo. We found that noscapine showed greaterthan 78% inhibition of the growth of intracranial glioma inimmunocompromised mice. Histopathology of noscapine-treated brain tissue revealed a dramatically reduced number ofmalignant cells and an increase of dilated blood vessels sur-rounded by a layer of neoplastic cells. In contrast, we observeda massive infiltration of tumor growth and an absence of dilatedvessels in vehicle-treated animals. Although noscapine treat-ment resulted in a marked reduction of tumor cells and tumorinfiltration, there remains a significant need for additional treat-ments that will target residual infiltrated tumor cells. Additionalstudies are warranted to examine whether noscapine will proveequally efficient in the treatment of mice carrying human tumorsthat exhibit a slower mitotic rate or for the treatment of spon-taneously occurring gliomas in transgenic mice.

The use of any chemotherapeutic agent that affects micro-tubule structure or dynamics raises a concern of neurotoxicity,particularly in regard to the peripheral nervous system. Thesetypes of drugs are thought to disrupt normal axonal transport,leading to axonal degeneration and clinical symptoms of numb-ness and/or weakness (45). It is encouraging that doses ofnoscapine that show efficacy against brain tumors did not causeany overt pathological changes in the peripheral nervous system.Additionally, sensory neurites died only with prolonged expo-sure to high doses of noscapine. There are no human reports ofperipheral neuropathy with the use of low dose noscapine as anantitussive, however, the assessment of neurotoxicity in thesetting of treatment for brain tumors requires human clinicaltrials.

We observed blood vessel dilation in noscapine-treatedtissue in both the tumor-infiltrated and contralateral brain hemi-spheres. Additional studies are warranted to examine whetherdilation is present in other organs, to determine whether thedilation observed relates to the antitussive effects of noscapine,and to assess whether the effects observed constitute a clinicalrisk at the noscapine doses required to achieve antitumor effects.

Noscapine only affects microtubule dynamics (22) ratherthan changing the net equilibrium between the monomer and thepolymer distribution of tubulin within the cell (23). Normal cellswith intact checkpoint proteins could conceivably tolerate therelatively less disruptive microtubule effects of noscapine com-pared with other known antimitotic agents. Normal microtubulemorphology is retained in nontumor cells after noscapine expo-sure, and cells resume cell division upon noscapine removal.Upon in vivo treatment, noscapine levels rise only transiently inplasma; pharmacokinetic studies in mice and humans revealpeak concentration at 3 h after oral ingestion and a relatively fastclearance thereafter. This suggests that normal cells likely re-sume cell division after the noscapine concentration decreasesbelow the threshold level in a few hours (27). In support of thishypothesis, BrdUrd measurements in duodenum, spleen, andliver did not show changes in cell division rates between treatedand untreated animals (data not shown). These properties ofnoscapine might explain the absence of toxicity at sites of




normally dividing tissue or in peripheral nerves observed in ourstudy.

Noscapine should be further tested in humans to confirm apositive adverse event profile and to examine its ability toinhibit the growth of the subset of central nervous systemtumors that show rapid proliferation rates such as glioblastoma.Perhaps, this could best be achieved in conjunction with othertherapies because noscapine alone did not eradicate the tumortype tested in this study. In conclusion, noscapine was able tosignificantly reduce the growth of a very aggressive experimen-tal mouse glioma and therefore is a promising anticancer agentthat provides novel hope for the treatment of malignant gliomasthat have a less than 20% response rate to conventional chem-otherapy (3) and for which existing treatments are associatedwith debilitating toxic side effects (46).

ACKNOWLEDGMENTSWe thank Laura Brown, Ivana Bonaccorsi, the members of the Joshi

and Archer laboratories for experimental assistance, Dr. Dirk Dillehay,veterinary pathologist, for blindly evaluating animal tissue sections, and Dr.Daniel Brat for reviewing brain pathology.

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2004;10:5187-5201. Clin Cancer Res Jaren W. Landen, Vincent Hau, Mingshen Wang, et al. Glioblastoma GrowthNoscapine Crosses the Blood-Brain Barrier and Inhibits

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